Protein Synthesis: Stage 1: Aminoacyl-tRNA Synthetases Attach the Correct Amino Acids to Their tRNAs
During the first stage of protein synthesis, taking place in the cytosol, aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each en zyme is specific for one amino acid and one or more corresponding tRNAs. Most organisms have one aminoacyl tRNA synthetase for each amino acid. For amino acids with two or more corresponding tRNAs, the same en zyme usually aminoacylates all of them. The structures of all the aminoacyl-tRNA synthetases of E. coli have been determined. Researchers have divided them into two classes (Table 27–7) based on substantial differences in primary and tertiary structure and in reaction mechanism (Fig. 1); these two classes are the same in all organisms. There is no evidence for a common ancestor, and the bio logical, chemical, or evolutionary reasons for two en zyme classes for essentially identical processes remain obscure.
The reaction catalyzed by an aminoacyl-tRNA syn thetase is Amino acid +t RNA +ATP 
aminoacyl-tRNA +AMP+ PPi
This reaction occurs in two steps in the enzyme’s active site. In step 1 (Fig. 1) an enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP), forms when the carboxyl group of the amino acid reacts with the -phosphoryl group of ATP to form an anhydride linkage, with displacement of pyrophosphate. In the second step the aminoacyl group is transferred from enzyme-bound aminoacyl-AMP to its corresponding specific tRNA. The course of this second step depends on the class to which the enzyme belongs, as shown by pathways 2a and 2b in Figure 1. The resulting ester linkage between the amino acid and the tRNA (Fig.2) has a highly negative standard free energy of hydrolysis (ΔG=-29 kJ/mol). The pyrophosphate formed in the activation reaction undergoes hydrolysis to phosphate by inorganic pyrophosphatase. Thus, two high-energy phosphate bonds are ultimately expended for each amino acid molecule activated, rendering the overall reaction for amino acid activation essentially irreversible:
Amino acid+ tRNA+ ATP 
aminoacyl-tRNA AMP 2Pi ΔG ≈ 29 kJ/mol
Proofreading by Aminoacyl-tRNA Synthetases The amino acylation of tRNA accomplishes two ends: (1) activation of an amino acid for peptide bond formation and (2) attachment of the amino acid to an adaptor tRNA that en sures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attach ment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. As you will recall from Chapter 6, enzyme specificity is limited by the binding energy available from en zyme-substrate interactions. Discrimination between two similar amino acid substrates has been studied in detail in the case of Ile-tRNA synthetase, which distinguishes between valine and isoleucine, amino acids that differ by only a single methylene group (-CH2-). Ile tRNA synthetase favors activation of isoleucine (to form Ile-AMP) over valine by a factor of 200—as we would expect, given the amount by which a methylene group (in Ile) could enhance substrate binding. Yet valine is erroneously incorporated into proteins in positions normally occupied by an Ile residue at a frequency of only about 1 in 3,000. How is this greater than tenfold increase in accuracy brought about? Ile-tRNA synthetase, like some other aminoacyl-tRNA synthetases, has a proofreading function. Recall a general principle from the discussion of proofreading by DNA polymerases : if available binding interactions do not provide sufficient discrimination between two substrates, the necessary specificity can be achieved by substrate-specific binding in two successive steps. The effect of forcing the system through two successive filters is multiplicative. In the case of Ile-tRNA synthetase, the first filter is the initial binding of the amino acid to the enzyme and its activation to aminoacyl-AMP. The second is the binding of any incorrect aminoacyl-AMP products to a separate active site on the enzyme; a substrate that binds in this second active site is hydrolyzed. The R group of valine is slightly smaller than that of isoleucine, so Val-AMP fits the hydrolytic (proofreading) site of the Ile-tRNA syn thetase but Ile-AMP does not. Thus Val-AMP is hydrolyzed to valine and AMP in the proofreading active site, and tRNA bound to the synthetase does not become aminoacylated to the wrong amino acid.
MECHANISM FIGURE 1 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step 1 is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27–7). For class I enzymes, 2a the aminoacyl group is transferred initially to the 2-hydroxyl group of the 3-terminal A residue, then 3ato the 3-hydroxyl group by a transesterification reaction. For class II enzymes, 2bthe aminoacyl group is transferred directly to the 3-hydroxyl group of the terminal adenylate.
FIGURE 2 General structure of aminoacyl-tRNAs. The amino acyl group is esterified to the 3position of the terminal A residue. The ester linkage that both activates the amino acid and joins it to the tRNA is shaded pink.
In addition to proofreading after formation of the aminoacyl-AMP intermediate, most aminoacyl-tRNA synthetases can also hydrolyze the ester linkage be tween amino acids and tRNAs in the aminoacyl-tRNAs. This hydrolysis is greatly accelerated for incorrectly charged tRNAs, providing yet a third filter to enhance the fidelity of the overall process. The few aminoacyl tRNA synthetases that activate amino acids with no close structural relatives (Cys-tRNA synthetase, for ex ample) demonstrate little or no proofreading activity; in these cases, the active site for aminoacylation can sufficiently discriminate between the proper substrate and any incorrect amino acid. The overall error rate of protein synthesis (~1 mis take per 104 amino acids incorporated) is not nearly as low as that of DNA replication. Because flaws in a protein are eliminated when the protein is degraded and are not passed on to future generations, they have less biological significance. The degree of fidelity in protein synthesis is sufficient to ensure that most proteins contain no mistakes and that the large amount of energy required to synthesize a protein is rarely wasted. One defective protein molecule is usually unimportant when many correct copies of the same protein are present. Interaction between an Aminoacyl-tRNA Synthetase and a tRNA: A “Second Genetic Code” An individual aminoacyl-tRNA synthetase must be specific not only for a single amino acid but for certain tRNAs as well. Discriminating among dozens of tRNAs is just as important for the overall fidelity of protein biosynthesis as is distinguishing among amino acids. The interaction between aminoacyl-tRNA synthetases and tRNAs has been referred to as the “second genetic code,” reflecting its critical role in maintaining the accuracy of protein synthesis. The “coding” rules appear to be more complex than those in the “first” code. Figure 3 summarizes what we know about the nucleotides involved in recognition by some aminoacyl tRNA synthetases. Some nucleotides are conserved in all tRNAs and therefore cannot be used for discrimination.
FIGURE 3 Nucleotide positions in tRNAs that are recognized by aminoacyl-tRNA synthetases. Some positions (blue dots) are the same in all tRNAs and therefore cannot be used to discriminate one from another. Other positions are known recognition points for one (orange) or more (green) aminoacyl-tRNA synthetases. Structural features other than sequence are important for recognition by some of the synthetases.
FIGURE 4 Aminoacyl-tRNA synthetases. Both synthetases are complexed with their cognate tRNAs (green stick structures). Bound ATP (red) pinpoints the active site near the end of the aminoacyl arm.
(a) Gln-tRNA synthetase from E. coli, a typical monomeric type I syn thetase (PDB ID 1QRT). (b) Asp-tRNA synthetase from yeast, a typical dimeric type II synthetase (PDB ID 1ASZ).
By observing changes in nucleotides that alter substrate specificity, researchers have identified nucleotide positions that are involved in discrimination by the amino acyl-tRNA synthetases. These nucleotide positions seem to be concentrated in the amino acid arm and the anti-codon arm, including the nucleotides of the anticodon itself, but are also located in other parts of the tRNA molecule. Determination of the crystal structures of aminoacyl-tRNA synthetases complexed with their cognate tRNAs and ATP has added a great deal to our un derstanding of these interactions (Fig. 4). Ten or more specific nucleotides may be involved in recognition of a tRNA by its specific aminoacyl-tRNA synthetase. But in a few cases the recognition mechanism is quite simple. Across a range of organisms from bacteria to humans, the primary determinant of tRNA recognition by the Ala-tRNA synthetases is a single GUU base pair in the amino acid arm of tRNAAla . A short RNA with as few as 7 bp arranged in a simple hairpin minihelix is efficiently aminoacylated by the Ala-tRNA synthetase, as long as the RNA contains the critical G=U . This relatively simple alanine system may be an evolutionary relic of a period when RNA oligonucleotides, ancestors to tRNA, were aminoacylated in a primitive system for protein synthesis.
